Molecular identification and functional characterization of a transcription factor GeRAV1 from Gelsemium elegans

Background Gelsemium elegans is a traditional Chinese medicinal plant and temperature is one of the key factors affecting its growth. RAV (related to ABI3/VP1) transcription factor plays multiple roles in higher plants, including the regulation of plant growth, development, and stress response. However, RAV transcription factor in G. elegans has not been reported. Results In this study, three novel GeRAV genes (GeRAV1-GeRAV3) were identified from the transcriptome of G. elegans under low temperature stress. Phylogenetic analysis showed that GeRAV1-GeRAV3 proteins were clustered into groups II, IV, and V, respectively. RNA-sequencing (RNA-seq) and real-time quantitative PCR (qRT-PCR) analyses indicated that the expression of GeRAV1 and GeRAV2 was increased in response to cold stress. Furthermore, the GeRAV1 gene was successfully cloned from G. elegans leaf. It encoded a hydrophilic, unstable, and non-secretory protein that contained both AP2 and B3 domains. The amino acid sequence of GeRAV1 protein shared a high similarity of 81.97% with Camptotheca acuminata CaRAV. Subcellular localization and transcriptional self-activation experiments demonstrated that GeRAV1 was a nucleoprotein without self-activating activity. The GeRAV1 gene was constitutively expressed in the leaves, stems, and roots of the G. elegans, with the highest expression levels in roots. In addition, the expression of the GeRAV1 gene was rapidly up-regulated under abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA) stresses, suggesting that it may be involved in hormonal signaling pathways. Moreover, GeRAV1 conferred improved cold and sodium chloride tolerance in Escherichia coli Rosetta cells. Conclusions These findings provided a foundation for further understanding on the function and regulatory mechanism of the GeRAV1 gene in response to low-temperature stress in G. elegans. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-023-09919-9.


Background
Under low temperature stress, plants undergo significant alterations in morphology, physiology, molecular biology, and metabolism, leading to functional disruption and impairments in growth and development [1].Transcription factors (TFs) are the major regulators in response to and coping with cold stress in plants [2].The primary signal transduction pathways were activated in plants under low temperature stress depend on inducer of C-repeat binding factor expression/dehydration response elements binding protein (ICE-CBF/DREB) [3].Besides, TFs like WRKY, leucine zipper (bZIP), v-myb avian myeloblastosis viral oncogene homolog (MYB), and NAC (NAM, ATAF1/2, CUC2) are capable of regulating gene expression and activating cold-regulated genes (CORs) [3].The APETALA2/ethylene-responsive factor (AP2/ERF) family, which can be categorized into subfamilies including AP2, ERF, RAV, and Soloist, is one of the most extensive groups of TFs in plants [4].Among them, RAV TFs, which not only contain AP2 domains but also have a B3 domain, are the specific class of TFs in higher plants, and are also classified as a B3 superfamily.The AP2 domain consisting of about 60 amino acids, which can recognize the CAACA motif, is located at the N-terminus of the RAV protein [4,5].Conversely, the B3 domain consisting of about 110 amino acids, which can recognize the CACCTG motif, is located in the conserved region at the C-terminus of the RAV protein [6].Interesting, the presence of both domains in RAV TFs can enhance the specificity and affinity of binding to targeted DNA for controlling gene expression [4,5].
Not surprisingly, in the AP2/ERF family, RAV TFs containing both AP2 and B3 domains have received increasing attention.In 1999, two novel RAV TFs (RAV1 and RAV2) have been identified for the first time from Arabidopsis thaliana [5].Subsequent study has proved that there are 13 RAV TFs in A. thaliana, six of which contain both AP2 and B3 domains, including AtRAV1 (At1g13260), AtTEM1 (At1g25560), AtTEM2 (At1g68840), At3g25730, At1g50680, and At1g51120 [6].The RAV family genes involved in plant growth and development and response to various stresses have been successively reported [7].Overexpression of Zea mays ZmRAV1 can enhance salt and osmotic tolerance in transgenic A. thaliana [8].The promoter region of the RAV subfamily contains cis-acting elements involving low temperature response (LTR), and can thus regulate gene expression under cold stress [9].Early cold stress rapidly induces overexpression of the BcRAV gene in Brassica campestris and improves its cold resistance [10].The VaRAV1 gene in Vitis amurensis plays a significant role in cold stress by regulating related TFs and inducing the expression of related genes in the cell wall [11].To sum up, one hand, RAV IFs can regulate plant seed development, root growth, flower opening, and leaf senescence [12][13][14][15].On the other hand, RAV IFs play important roles in biotic and abiotic stresses in plants, including disease, cold, drought, and salt resistance [8,10,16].
Gelsemium elegans (Gardner and Champ.)Benth., the medicinal vine plant commonly known as Gelsemium or "heartbroken grass", was first recorded in the traditional Chinese medicinal book "Shennong Materia Media".Although the entire plant of G. elegans is highly toxic, it has a high medicinal value [17].The effective active components of G. elegans were indole alkaloids.Over a hundred different types of monoterpenoid indole alkaloids have been extracted from the roots [18], stems, leaves [19], fruits [20], and other organs of G. elegans, which provide a valuable reference for the development of new drugs [21,22].Researches have shown that the alkaloids in G. elegans are pharmacologically active in pain relief [23], anti-inflammatory effects [24], immunomodulatory [25], and therapeutic for depression and anxiety [26].Surprisingly, nearly half of the common drugs come from natural products, especially alkaloids, making alkaloid-rich medicinal plants the center of attention [27].G. elegans mainly grows in Southeast Asia and southern China [28].Low temperature can affect the growth, metabolite biosynthesis and gene expression of medicinal plants, such as Angelica sinensis [29], Dysosma versipellis (Hance) M. Chang [30], and Podophyllum hexandrum Royle [31].Similary, G. elegans cannot tolerate low temperatures during its growth.Prolonged exposure to low temperatures leads to morphological and physiological changes in G. elegans, which may affect the content and activity of its active ingredients [32].
Currently, there is still no report on the role of RAV TFs from G. elegans in cold tolerance.In this study, three RAV subfamily genes were identified and analyzed from the G. elegans transcriptome database constructed by our research group under low temperature stress (unpublished).Based on the transcriptome data and qRT-PCR analysis, one G. elegans GeRAV1 gene significantly induced by 4°C low temperature was successfully cloned.Besides, the sequence characteristics, protein properties, structural domains, phylogenetic tree, subcellular localization, and self-activation activity of the GeRAV1 gene were analyzed.Its expression in different G. elegans tissues and hormone treatments was detected by qRT-PCR technique.Moreover, the tolerance of GeRAV1 to abiotic stress was investigated using prokaryotic expression systems.The findings of this study should serve as a basis for further exploration of the function and regulatory mechanism of RAV TFs in the response of G. elegans to low-temperature stress.

Identification and sequence analysis of G. elegans RAV subfamily genes
Three GeRAV genes (GeRAV1, c23896.graph_c0;GeRAV2, c34018.graph_c1;and GeRAV3, Gec35851.graph_c0) were identified from the transcriptome of G. elegans under 4°C low temperature stress which was previously constructed by our research group.According to those reports on the whole-genome analysis of the RAV gene family [33][34][35], a phylogenetic tree was constructed using the protein sequences of the GeRAV proteins and RAVs from two dicot plants (A.thaliana and Glycine max) and three monocot plants (Z.mays, Oryza sativa, and Triticum aestivum) (Fig. 1).A total of 77 RAV proteins were divided into five groups (Group I-V).Interestingly, GeRAV1 was clustered into Group II with proteins containing AP2 and B3 domains, and the other two GeR-AVs (GeRAV2 and GeRAV3) were clustered into two respective groups (Group IV and Group V) containing only B3 domains.A total of ten conserved motifs were predicted for RAV proteins using the MEME website and 37.7% of RAV proteins contained more than nine motifs (Fig. 2).From the N-terminus to the C-terminus, all 77 RAV proteins contained motifs 3, 2, and 1, suggesting Fig. 1 Phylogenetic tree of GeRAVs and other plant RAV proteins.The RAVs used in the phylogenetic tree analysis were from three dicot species (Gelsemium elegans, Glycine max, and Arabidopsis thaliana) and three monocot species (Triticum aestivum, Oryza sativa, and Zea mays) [33][34][35].The GenBank accession numbers of all RAV homologous proteins were listed in Table S5.Groups I-V were marked in blue, pink, green, purple, and orange, respectively that they were relatively conserved in RAV genes evolution.Besides, in terms of conserved domains, all RAV proteins have B3 domains at the C-terminus, but do not always contain both AP2 and B3 domains (Fig. 2).In addition, the proteins in the groups I, II, and III had both AP2 and B3 domains (Fig. 2).

The expression levels of GeRAV genes in response to low temperature stress
The expression profiles of the three GeRAV genes in G. elegans were analyzed under 4°C cold stress based on our transcriptomic data.The results showed that the expression of GeRAV1 and GeRAV2 were up-regulated under 4°C cold stress, whereas GeRAV3 was down-regulated.GeRAV1 exhibiting the highest expression profiles at 6 and 12 h (Fig. 3A).The qRT-PCR validation also showed that, under cold stress, the expression of GeRAV1 gene maintained at a significant up-regulated level at 6, 12 and 24 h, which was about 4.75, 3.84 and 3.23 times higher than the control, respectively (Fig. 3B).Similarly, the expression of GeRAV2 was also increased from 6 to 24 h, which was about 1.44, 1.77 and 1.67 times higher than the control, respectively (Fig. 3B).However, there was no significant change in the expression levels of the GeRAV3 gene (Fig. 3B).These findings indicated that the GeRAV1 and GeRAV2 genes were induced by cold stress with upregulated expression.

Cloning and bioinformatics analysis of GeRAV1
As the GeRAV1 gene was significantly up-regulated by low temperature stress (Fig. 3), its cDNA sequence (1272 bp) was amplified by RT-PCR using the RNA extracted from the leaves of G. elegans as a template.The GeRAV1 gene contained an open reading frame (ORF) of 1083 bp, encoding a protein of 360 amino acids (Fig. S1) with a relative molecular weight of 40 kDa.The theoretical isoelectric point, the average hydrophilicity, and the instability coefficient of the GeRAV1 protein was 9.12, -0.554, and 40.03, respectively, suggesting that it was an unstable and hydrophilic protein.The absence of a signaling peptide or transmembrane region in the GeRAV1 protein indicated that it was a non-secretory protein.Secondary structure prediction results showed that the GeRAV1 protein was mainly composed of a random coil (50.28%), alpha helix (23.89%), extended chain (19.72%), and beta-turn (6.11%) (Fig. S2).
Comparative analysis of the predicted tertiary structures revealed that the GeRAV1 protein of G. elegans Fig. 2 Phylogenetic tree, conserved motif, and conserved domain analyses of GeRAV1 and other species RAVs.Phylogenetic tree: the phylogenetic tree was the same as the evolutionary tree in Fig. 1.Conserved motif: the 10 conserved motifs corresponded to ten boxes of different colors.Conserved domains: B3 domains and AP2 domains were shown as pink and blue boxes, respectively.Motif logos: ten motif logo patterns and sequences were shown in Table S6 was highly similar to the RAV1 proteins of A. thaliana (At1g13260) and G. max (Glyma.01G22260).These three proteins contained an AP2 domain that consisted of an α-helix and a β-barrel protein, and the β-barrel protein was located parallel to the α-helix.In addition, the B3 domain of GeRAV1 contained two α-helices and one β-barrel protein, where the α-helices protruded from both ends of the β-barrel protein (Fig. 4).The amino acid sequence similarity of GeRAV1 with Camptotheca acuminata CaRAV (QNI23763.1),Olea europaea OeRAV1 (CAA2976529.1),Vitis vinifera VvRAV1 (XP_002281709.2),Carya illinoinensis CiRAV1-like (XP_042963336.1),G. max GmRAV-03 (Glyma.02G11060),and A. thaliana AtRAV1 was 81.97%, 79.95%, 74.55%, 76.55%, 59.31%, and 60.50%, respectively (Fig. S3).Notably, their amino acid sequence of the AP2 and B3 domains were highly conserved.The amino acid sequence of GeRAV1 showed certain conservation, with the N-terminal AP2 domain containing three β-strands (forming a β-barrel protein) and one α-helix, and the C-terminal B3 domain containing seven β-strands (forming a β-barrel protein) and two α-helices (α1 and α2) (Fig. S3).Furthermore, the predicted nuclear localization sequence (NLS) of the GeRAV1 protein was located at the C-terminus (Fig. S3).

Relative expression of GeRAV1 gene in G. elegans tissues and under hormonal stress
Expression levels of GeRAV1 in the tissues of G. elegans were determined using the qRT-PCR technique.The results showed that the GeRAV1 gene was constitutively expressed in different tissues of G. elegans, with the highest expression levels in the roots, which was approximately 3.6 and 4.38 times higher than those in the leaves and stems, respectively (Fig. 5A).In addition, the expression of GeRAV1 was induced by treatments with salicylic acid (SA), abscisic acid (ABA), and methyl jasmonate (MeJA) (Fig. 5B, C, D).GeRAV1 reached peak expression levels at 6 h under SA stress and at 3 h under both ABA and MeJA stimulus.

GeRAV1 was localized to the nucleus
Subcellular localization of the GeRAV1 protein in the nucleus was predicted using WoLF PSORT and Plant-mPLoc online servers.To confirm this prediction, 35S::GeRAV1::GFP was used as an experimental group and 35S::GFP was used as a control group by Agrobacterium-mediated injections in Nicotiana benthamiana leaves.Confocal microscopy did not observe fluorescence signals in the control leaf cells, while the experimental group showed green fluorescence in the nucleus of leaf cells.Additionally, NbH2B (histone H2B) displayed red fluorescence in the nucleus of leaf cells.The overlap of green and red fluorescence resulted in yellow fluorescence, providing further evidence for the subcellular localization of the GeRAV1 protein in the nucleus (Fig. 6).

GeRAV1 had no self-activating activity
The presence of colonies on the tryptophan-deficient medium (SDO) plate of pGBKT7-GeRAV1, pGBKT7-p53 + pGADT7-T (positive control), and pGBKT7 (negative control) indicated the successful transformation of recombinant plasmids into Y2HGold yeast (Fig. 7).The growth of colonies showed a consistent trend with the gradient of diluted liquid culture, indicating that the GeRAV1 protein bound to GAL4-BD and expressed the missing tryptophan in the medium.Colonies of pGBKT7-GeRAV were observed on the SDO/X-α-gal (5-bromo-4-chloro-3-indole-α-dgalactoside) (SDO/X) plate, as well as positive and negative controls.However, upon the addition of X-α-gal, only the positive control displayed blue color due to the activation of the MEL1 reporter gene, while the rest of the samples showed no color change, suggesting that the GeRAV1 protein had no self-activation activity.On the SDO/X/AbA (aureobasidin) (SDO/X/A) plate, only the positive control formed blue colonies, indicating activation of both AUR1-C and MEL1 reporter genes.In contrast, no colonies were observed for the negative control and pGBKT7-GeRAV1 (Fig. 7), further confirming the absence of transcriptional self-activation activity in the GeRAV1 protein.

Stress tolerance assays of the GeRAV1 gene in Escherichia coli Rosetta (DE3)
The GeRAV1 protein has a predicted relative molecular weight of 40 kDa.pET32a expression vector contains a 6×His tag [36].When recombinant cells (pET32a-GeRAV1-Rosetta) were successfully induced under specific conditions of 16°C, 28°C, 37°C, and 0.7 mM isopropyl β-D-thiogalactoside (IPTG), an obviously accumulated protein was observed above 40 kDa (Fig. 8A).As shown in Fig. 8B, the recombinant cells (pET32a-GeRAV1-Rosetta) had similar growth conditions as the control cells (pET32a-Rosetta) with mannitol supplement.However, the growth of recombinant cells (pET32a-GeRAV1-Rosetta) was significantly better than that of the control cells (pET32a-Rosetta) when treated with low temperature treatment at 4°C and sodium chloride (Fig. 8B).These results suggested that overexpression Fig. 7 Transcriptional self-activation status of the GeRAV1 protein in yeast.SDO: Synthetic defined medium without tryptophan; SDO/X: synthetic defined medium without tryptophan supplemented with X-α-Gal; SDO/X/A: synthetic defined medium without tryptophan supplemented with X-α-Gal and aureobasidin A. pGBKT7-p53 + pGADT7-T: Positive control; pGBKT7: negative control

Discussion
Plants have evolved a molecular regulatory network and cold resistance mechanisms to cope with low temperature stress.It has been found that plant cells sense changes in temperature upon low-temperature stimulation, which triggers signaling receptors, such as Ca 2+ permeation channels, receptor kinases, histidine kinases.[1,37].Subsequently, led by substances such as Ca 2+ , ABA, and reactive oxygen species, the low temperature signal was transmitted downward to activate TFs and regulate the expression of downstream CORs to enhance cold resistance [1,37].Within the AP2/ERF superfamily, the CBF/DREB TFs have been extensively and comprehensively studied in response to cold stress, as the CBF proteins can act on cis-acting elements and induce the expression of downstream CORs [1].Recent research has identified RAV TFs belonging to the AP2/ERF family as cold-responsive TFs that may trigger regulatory pathways distinct from the CBF cold-responsive pathway [38].Notably, RAV1 is a TF independent of the CBF pathway and plays a vital role in cold treatment and cold domestication [39].Currently, RAV TFs respond positively to low temperature stress and have been cloned and studied in various plants such as A. thaliana [9], O. sativa [40], Camellia sinensis [41], Galegae orientalis [42], and V. amurensis [11].

Sequences and phylogenetic characterization of GeRAV1
In this study, a GeRAV1 gene with a significant response to low temperature stress was identified and characterized in the G. elegans.Tertiary structure prediction and multi-sequence alignments showed that the GeRAV1 protein exhibited similar structural features to AtRAV1 in A. thaliana (Fig. 4, Fig. S3), including the same number and the similar positions of α-helices and β-sheets in the AP2 and B3 domains, that is, as well as other reported RAVs containing the AP2 and B3 domains [6,33,43].In addition, the AP2 and B3 domains showed a high degree of conservation between different amino acid sequences of RAVs from various plant species (Fig. S3), suggesting their important role in DNA binding specificity [44].Interestingly, we also found the highest homology of 81.97% between G. elegans GeRAV1 and C. acuminata CaRAV (QNI23763.1)(Fig. S3).Therefore, it is hypothesized that there is a closer relationship between G. elegans and other genes in C. acuminate, which is conducive to our future research on alkaloid-related genes of G. elegans.Besides, GeRAV1 clustered closely with AtRAV1, At3g25730, AtTEM1, AtTEM2, GmRAV-03, GmRAV-01, GmRAV-08, and GmRAV-13 (Fig. 1), indicating that there is a closer evolutionary relationship between GeRAV1 and two dicotyledonous plants (A.thaliana and G. max) than the other three monocotyledonous plants (Z.mays, O. sativa, and T. aestivum).GmRAV-03 has been reported to enhance NaCl and drought tolerance in transgenic A. thaliana, but is insensitive to exogenous ABA [33]. A. thaliana AtRAV1 has been found to be involved in the cold response [9].GeRAV1 belonged to the same group as GmRAV-03 and AtRAV1, with similar sequence type, number, and motif position (Fig. 2), indicating a similar biological function in the cold response.Previous studies have shown that the AP2 domain is a plant-specific domain involved in meristematic organization, flowering, seed development, and stress response [45].The B3 domain binds to specific DNA and regulates plant growth, development, and hormone signaling [6,46].Considering that the GeRAV1 protein contained both AP2 and B3 domains, it is possible that GeRAV1 may not only play a regulatory role in cold stress but also affect the growth and development of G. elegans and participate in certain hormone signaling pathways.

GeRAV1 gene was constitutively expressed in various tissues and responded positively to cold stress
Differential expression of the GhRAV gene has been observed in roots, stems, and leaves of Gossypium hirsutum, with a more significant increase in expression in roots and stems [47].We found that the GeRAV1 gene in G. elegans was expressed in root, stem, and leaf tissues, with a higher expression level in roots (Fig. 5A).G. elegans roots have higher levels and types of alkaloids than leaves and stems [28,48], which was consistent with previous findings that over half of the extracted monoterpene indole alkaloids come from roots [22].In the present study, we confirmed that the GeRAV1 gene was highly expressed in G. elegans roots, does this imply a potential role in regulating the synthesis and accumulation of alkaloids in the roots of G. elegans?
As reported, the RAV genes exhibited a response to cold stress [38].In maize seedlings, the ZmRAV3 gene was significantly overexpressed after treatment at 4°C for 2 h [49].The expression of the BraRAV-14 gene in Chinese cabbage was up-regulated under low temperature at 4°C, peaking at 24 h [50].The RAV family genes in Brassica napus showed up-regulated expression at four time points (2,6,12, and 24 h), with nearly half of the RAV genes reaching their highest expression levels at 6 h [10].Based on the transcriptome data and qRT-PCR analysis, three GeRAV genes were differentially expressed in G. elegans at 4°C (Fig. 3).Interestingly, the expression levels of the GeRAV1 gene were significantly up-regulated from 6 to 24 h under 4°C treatment.In addition, prokaryotic expression analysis showed that GeRAV1 enhanced the tolerance of E. coli cells to low temperature (4°C) and NaCl treatments (Fig. 8B).These results indicating the potential role of the GeRAV gene in the response of G. elegans to low temperature and salt stresses.Similarly, previous studies have shown that RAV genes improved the tolerance of plants under NaCl and drought stresses [51,52].

Responses of GeRAV1 to phytohormones stress
As one of the important signaling compounds, phytohormones can mediate plant response to abiotic stress [53].The expression of the GeRAV1 gene was significantly up-regulated at 3, 6 and 12 h under SA treatment and at 3 h under MeJA treatment (Fig. 5B, D), suggesting that the GeRAV1 gene could be induced by SA and MeJA hormone stresses.Previous studies also showed that the expression of most OsRAVs in rice was upregulated at 12 h under the treatments of MeJA and SA [35].After challenging by ABA, the expression levels of MtRAVs, GhRAV1, and GmRAV-03 were increased in Medicago truncatula, Gossypium herbaceum, and G. max [33,[54][55][56].However, transgenic A. thaliana plants with the MtRAVs, GhRAV1, and GmRAV-03 genes were insensitive to exogenous ABA, suggesting that these RAV genes might be participate in the ABA signaling pathway through an ABA-independent mechanism [33,[54][55][56].In cucumber, CsABI5 (ABA-associated marker gene) was significantly induced under ABA treatment and the transgenic Arabidopsis overexpressing CsRAV1 was more tolerant to ABA, indicating that CsRAV1 may directly regulate the expression of CsABI5, thereby participating in the ABA signaling pathway [57].In this work, the GeRAV1 gene was induced at 3, 6, and 12 h of ABA treatment (Fig. 5C).These results indicate that the GeRAV1 gene of G. elegans may play a role in hormonal response.Further verification is needed to determine whether the GeRAV1 gene is also involved in the ABA signaling pathway through an ABA-independent mechanism.

Subcellular localization and transcriptional activation of GeRAV1 protein
In plants, RAV TFs were involved in regulating biosynthesis and metabolism.Zhao et al. demonstrated that the NnRAV1 gene could interact with the NnbHLH1 gene to regulate the synthesis and accumulation of alkaloids in Nelumbo nucifera [58].In Solanum tuberosum, the StRAV1 gene regulated anthocyanin accumulation in tubers by repressing the expression of key anthocyanin biosynthesis structural genes such as StCHS, StANS, and St3'5'H by suppressing their promoters [59].
McRAV1 acted as an inhibitor of anthocyanin biosynthesis in Malus crabapple [60].Similarly, in M. esculenta, the CAACA motif in the RAV TFs was common in the promoters of melatonin biosynthesis-related genes.MeRAV1 and MeRAV2, as upstream TFs, directly activated three melatonin biosynthesis genes in cassava [61].
The FaRAV1 gene directly bound and activated the promoters in the anthocyanin biosynthetic pathway that promoted anthocyanin accumulation in Fragaria ananassa [62].Previous research demonstrated that anthocyanins confer a variety of colors to organs such as flowers and fruits, and play important roles in resisting stressors like low temperature and bright light [63].RAV TFs in various plant species have been reported to function as nucleoproteins, such as G. max GmRAV-01 [64], G. orientalis GoRAV [42], Manihot esculenta MeRAV1/2 [65], M. truncatula MtRAV1/2/3 [54], A. thaliana AtRAV1 [12], Betula platyphylla BpRAV1 [66], Capsicum annuume CaRAV1 [16], G. herbaceum GhRAV1 [56], and Z. mays ZmRAV1 [8].Similarly, the subcellular localization results indicated that the GeRAV1 was a nuclear-localized protein (Fig. 6).It is worth noting that ZmRAV1 in Z. mays and TaRAV1 in T. aestivum displayed transcriptional activity in yeast cells [8,34], whereas NtRAV4 in Nicotiana tabacum did not [67].In this study, the GeRAV1 gene has no self-activation activity (Fig. 7), proving that it can be used as a bait, laying the foundation for mining functional genes that interact with it.This study will contribute to our further understanding of the biological function of the G. elegans GeRAV1 gene.However, the role of RAV TFs in regulating biosynthesis and secondary metabolites in G. elegans still needs further investigation.

Conclusion
In the present study, three novel RAV subfamily genes, whose encoding proteins were divided into three groups, were mined from the G. elegans transcriptome database under low temperature stress.Besides, the full-length GeRAV1 gene, which was significantly induced by 4°C low temperature, was cloned and characterized.GeRAV1 was a nucleoprotein without self-activating activity.The expression of GeRAV1 was observed in all the G. elegans tissues and the transcripts mainly accumulated in roots.Furthermore, GeRAV1 mRNA expression was up-regulated under SA, MeJA, and ABA stimulus.In particularly, overexpression of GeRAV1 could enhance the tolerance of E. coli Rosetta cells to low temperature and NaCl stresses, indicating that it is a potential candidate gene for genetic manipulation to improve low temperature and salt tolerance in G. elegans.These findings provide a basis for studying the biological functions and molecular regulatory mechanisms of RAV TFs in G. elegans.

G. elegans materials and treatments
The G. elegans plants were derived from Yongding District, Fujian Province, China (at an altitude of 602 m, coordinates: 116.9°E, 24.4°N).These plant materials were identified by Professor Zhongyi Zhang from Fujian Agriculture and Forestry University as an evergreen woody vine plant belonging to the Loganiaceae family and Gelsemium genus.The roots, stems, and leaves of G. elegans were gathered and rapidly frozen in liquid nitrogen prior, and then stored in a -80°C freezer.Meanwhile, G. elegans seedlings were cultivated in a laboratory greenhouse under controlled conditions (25°C, 14 h of light, and 23°C,10 h of darkness).Subsequently, the G. elegans plants with uniform growth were selected and subjected to the following four experiments.Under 4°C low temperature treatment, leaf samples were collected at 0, 6, 12, and 24 h [68].The leaves of G. elegans were subjected to a foliar spray of 100 µM MeJA, 5 mM SA, and 100 µM ABA [69,70], with leaf samples being collected at 0, 3, 6, 12, 24, and 48 h [70,71].

Total RNA extraction and the first-strand cDNA synthesis
The G. elegans samples were ground to extract total RNA using the TRIzol method (Invitrogen, USA).According to the Hifair® cDNA Synthesis Kit (Yeasen, Shanghai, China), RNA from G. elegans leaves was reverse-transcribed into cDNA for subsequent gene cloning experiments.Based on the Hifair® cDNA Synthesis SuperMix kit (Yeasen, Shanghai, China), RNA of G. elegans tissues and G. elegans samples under different treatments was reverse-transcribed into cDNA and used as a template for qRT-PCR analysis.

Mining GeRAV genes from G. elegans
Based on the transcriptome database of G. elegans under 4°C treatment, three GeRAV genes, namely GeRAV1 (c23896.graph_c0),GeRAV2 (c34018.graph_c1), and GeRAV3 (c35851.graph_c0),were identified.The amino acid sequences of the RAV family members from A. thaliana, O. sativa, Z. mays, G. max, and T. aestivum were downloaded from TAIR, Phytozome, and Ensembl Plants databases [33][34][35] and were used for phylogenetic tree and conserved structural domains and motifs analyses with the three GeRAV proteins.Clustal X was used to perform multiple sequence comparisons.Evolutionary tree was constructed using MEGA X (Neighbor-Joining method, Bootstrap = 1000) [72].EvolView was employed to beautify the evolutionary tree [73].For conserved motifs analysis, the number of motifs was set to 10 with other parameters remaining at their default values by MEME website [74].The prediction of conserved domains was carried out using NCBI-CDD (Table S1).TBtools was used to visualize the outcomes of the evolutionary tree, conserved domains, and conserved motifs [75].

Cloning of GeRAV1 gene and bioinformatics analysis
Based on the unigene sequence of the GeRAV1 gene which has significant expression levels under low temperature stress, its cloning primers (Table S2, Table S3) were designed using Primer 5.0.The PCR products were ligated into the Blunt-Zero vector (Yeasen, Shanghai, China).The GeRAV1 ORF sequence was analyzed using the ORF-finder program.The physicochemical properties, signal peptides, and transmembrane regions of the GeRAV1 protein were predicted using online software tools ProtParam, Net-Phos 3.1 server, SignalP 4.1 server, and TMHMM-2.0,respectively.The secondary structure of the protein was predicted using SOPMA.AlphaFold and PyMOL were used to predict and display the tertiary structure of the proteins, respectively [76].Homologous amino acid sequences of GeRAV1 were searched using NCBI-blastp online website.Sequence comparison of GeRAV1 with other RAVs from the Dicot GenBank database was performed using DNAMAN6.0 software.The NLS was predicted using the online tool cNLS Mapper.The information of bioinformatics tools used was listed in Table S1.

Expression patterns of the GeRAV genes in different tissues and under various stresses
The expression levels of the G. elegans GeRAV genes under low-temperature, MeJA, ABA, and SA treatments were examined using qRT-PCR technology.The details of designing quantitative primers for the GeRAV genes were shown in Table S3.The GeCUL gene of G. elegans, previously screened by our research group, was used as the reference gene (not yet published).qRT-PCR reaction system followed the instructions of the ChamQ Universal SYBR qPCR Master Mix (Nazyme, Nanjing, China).The qRT-PCR reaction was performed using the Quant-Studio™ 6 Flex real-time fluorescence quantitative PCR system.The melting and standard curves of the GeRAV genes were shown in Table S4.Each sample was performed in triplicate and sterile water was used as a control.Relative expression levels of the GeRAV1 genes were calculated using the 2 -ΔΔCT method [77] and data significance analysis was conducted using SPSS 19.0.0.Origin 2022 was used for data visualization and plotting.

Subcellular localization
GeRAV1 subcellular localization was predicted using WoLF PSORT and Plant-mPLoc server online tools (Table S1).Based on the gateway technology system, the primers Subloc-GeRAV1-F/R (Table S2) were designed.The positive plasmid pEASY-Blunt Zero-GeRAV1 obtained by cloning was served as a template for the BP reaction [78].The product was then ligated into the entry vector pDONR207 to produce the positive plasmid pDONR207-GeRAV1.Subsequently, an LR reaction (Invitrogen, USA) was performed to connect it to the subcellular localization vector pFAST-R05, which expresses a gene fused to the green fluorescent protein gene (GFP) that follows the ccdB gene (with a stop codon) [79].The positive recombinant plasmid pFAST-R05-GeRAV1-GFP (35S::GeRAV1::GFP) and the empty vector pFAST-R05-GFP (35S::GFP) were transformed into A. tumefaciens GV3101.After bacterial solution expansion and culture, the centrifuged culture was collected and washed twice with solution (10 mM magnesium chloride, 10 mM ethylmethylsulfone, pH = 5.0-5.4),then resuspended and adjusted OD 600 ≈ 0.6, followed by the addition of 200 µM acetosyringone.Post 2-3 h of induction under dark conditions, the solution was injected into N. benthamiana leaves of 5-6 leaf ages.A bacterial solution containing NbH2B (histone H2B) was also injected into the leaves of N. benthamiana [80].Subcellular localization of the GeRAV1 protein was observed using a Leica TCS SP8 confocal laser scanning microscope (Leica, Germany) after 2 days of incubation at 28°C and 16 h light/8 h dark.

Transcriptional self-activation
The self-activation activity of GeRAV1 was investigated using the Matchmaker Gold yeast two-hybrid system (with AUR1-C and MEL1 as reporter genes) [81].First, the DNA-binding domain (BD) vector pGBKT7 was linearized by double digestion with EcoRI and BamHI restriction enzymes.Primers pGBKT7-GeRAV1-F/R (Table S2), designed with specific restriction sites, were used for PCR amplification and gel purification.Then, the linearized vector and gel-purified target fragment were ligated for homologous recombination using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China).Finally, the recombinant plasmid pGBKT7-GeRAV1, BD-pGBKT7 vector (negative control), and hybridization vector plasmid pGBKT7-53 + pGADT7-T (positive control) were transformed into yeast Y2HGold competent cells (Weidi, Shanghai, China).After confirming the correctness of the cultures by PCR, the cultures were diluted to 1 × 10 -1 , 1 × 10 -2 , 1 × 10 -3 , and 1 × 10 -4 .Solid culture media including SDO, SDO/X, and SDO/X/A were prepared, and 7 µL of the diluted cultures were spotted onto the plates.All the plates were placed at 29°C for 2-3 d and photographed.

Expression of GeRAV1 gene in E. coli Rosetta (DE3) cells
According to the pET32a vector map, primers pET32a-GeRAV1-F/R (Table S2) were designed with restriction endonuclease sites BamHI and HindIII to obtain the recombinant plasmid pET32a-GeRAV1.Subsequently, the recombinant plasmid pET32a-GeRAV1 and the pET32a vector were transformed into E. coli Rosetta (DE3) cells (TIANGEN, Beijing, China).The E. coli Rosetta (DE3) cells containing pET32a-GeRAV1, pET32a vector, and no vector were each cultured in 20 mL centrifuge tubes, respectively, and their OD 600 was adjusted between 0.6 and 0.7 and induced with 0.7 mM IPTG at 16°C, 28°C, and 37°C.All collected bacterial cultures were centrifuged and then loaded with a loading buffer.After boiling treatment at 100 °C, samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis for gel running.Following that, coomassie blue fast staining solution (Beyotime, Shanghai, China) was performed.The gel was then destained to observe the target bands.Finally, the FUSION FX.EDGE SPECTRA multi-color fluorescence & chemiluminescence (VILBER BIO IMAGING, Paris, France) was used for imaging.
The method of plate stress was used to investigate the response of E. coli Rosetta cells containing the GeRAV1 gene under different stresses.When the bacterial cells (E. coli Rosetta (DE3) contains pET32a-GeRAV1 and pET32a vectors, respectively) reached OD 600 = 0.6, 0.7 mM IPTG was added to induce protein expression at 37°C.After 10 h of induction, the bacterial solution was adjusted to OD 600 = 0.6 and then diluted again to 10 − 3 and 10 − 4 .A total of 10 µL dilution was spotted on the corresponding plate, and then the stress tests of low temperature at 4°C (3, 6, 12 d), sodium chloride (150, 300, and 450 mM) and mannitol (300, 500, and 700 mM) were performed.Afterward, they were placed in a 37°C incubator overnight for the growth of bacterial colonies and then photographed [82].

Fig. 4 Fig. 3
Fig.4 Predicted tertiary structures of Gelsemium elegans GeRAV1, Arabidopsis thaliana AtRAV1, and Glycine max GmRAV-01 proteins.The positions of the AP2 domain and the B3 domain were respectively outlined in red dotted lines.The AP2 domain included an α-helix and a β-barrel protein, while the B3 domain included two α-helices (α1 and α2) and a β-barrel protein

Fig. 5
Fig. 5 Relative expression analysis of the GeRAV1 gene in different Gelsemium elegans tissues (A) and response to SA (B), ABA (C), and MeJA (D) stresses by qRT-PCR.GeCUL was used as the reference gene.Different lowercase letters on the bars indicated a significant difference, determined by Duncan's new multiple range test (P < 0.05).Data points represented mean ± standard error (n = 3)